Kris Chadee

Mucin and Toll-like receptors in host defense against intestinal parasites

Gastrointestinal mucin is a constituent of luminal barrier function and is the first line of host defense against invading pathogens. Mucin carbohydrates and amino acids, as well as trapped soluble host defense molecules, serve as substrates for colonization and control or deter pathogen invasion to the underlying mucosal epithelial cells. Toll-like receptors on the surface of epithelial cells act as sensors for invading pathogens, and the ensuing host response limits parasite invasion and leads to adaptive immunity. The latest work in the field and the use of parasite model systems to illustrate the delicate host-parasite interaction at the mucosal surface of the gut are discussed here.

Entamoeba histolytica Cysteine Proteinases Disrupt the Polymeric Structure of Colonic Mucin and Alter Its Protective Function

ABSTRACT The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [35S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time- and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.

DNA vaccines: designing strategies against parasitic infections

The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites.

Entamoeba histolytica stimulates interleukin 8 from human colonic epithelial cells without parasite-enterocyte contact

BACKGROUND & AIMSnThe mechanisms involved in the initiation of host mucosal inflammation in amebiasis are not fully understood. This study characterized the effect of Entamoeba histolytica components on interleukin 8 (IL-8) gene expression in human colonic cells.nnnMETHODSnColonic cells were stimulated with amebic proteins, secretory components, or live trophozoites (separated with 0.45-microm pores), and the levels of IL-8 messenger RNA (mRNA) and protein were detected.nnnRESULTSnLive amebae or their components enhanced IL-8 mRNA levels in the colonic cells (T84, LS174T, and Caco-2). In T84 cells, the accumulation of IL-8 mRNA induced by amebic components occurred in a dose- and time-dependent fashion. Increased secretion of IL-8 was noted after 12-hour stimulation; neutralizing antibodies against IL-15 or tumor necrosis factor alpha did not inhibit IL-8 production. Nuclear run-on assays showed that amebae-induced IL-8 gene occurred by a posttranscriptional mechanism. Cycloheximide treatment resulted in superinduction of IL-8 mRNA; however, dexamethasone inhibited E. histolytica-induced IL-8 gene expression.nnnCONCLUSIONSnE. histolytica can directly stimulate the induction of IL-8 by colonic cells in the absence of cell-cell contact or injury.

Prostaglandin E2 stimulates rat and human colonic mucin exocytosis via the EP4 receptor

Abstract Background & Aims: Mucins form an integral part of innate host defenses against intestinal pathogens and irritants. However, the mechanisms whereby mucin secretion is regulated during inflammation are poorly understood. Because prostaglandin E 2 (PGE 2 ) is prominent during intestinal inflammation, we investigated its receptor-signaling pathway coupled to mucin exocytosis in the colonic epithelial cell line LS174T and rat colon. Methods: Reverse-transcription polymerase chain reaction (RT-PCR) and [ 3 H]PGE 2 binding assays were used to identify the PGE 2 receptors (EP). Intracellular cyclic adenosine monophosphate ([cAMP] i ) was quantified by enzyme immunoassay. Mucins were metabolically labeled with [ 3 H]glucosamine, and mucin secretion was quantified by Sepharose 4B column chromatography, immunoblot analysis, and cesium chloride density gradient centrifugation. Results: RT-PCR and DNA sequence analysis identified EP 2 , EP 3 , and EP 4 receptors. Mucin secretion and [cAMP] i production by LS174T cells were stimulated dose-dependently by PGE 2 , the EP 4 -receptor agonist 1-OH-PGE 1 , and the EP 3 /EP 4 agonist MB117:1352-1362

Interaction of LS174T Human Colon Cancer Cell Mucins With Entamoeba histolytica: An In Vitro Model for Colonic Disease

BACKGROUND & AIMSnColonic mucins secreted by goblet cells protect the colon by preventing the attachment of enteric pathogens to the epithelium. Entamoeba histolytica overcomes this protective barrier and causes ulcerations, allowing the parasite to disseminate to the liver and form abscesses. An in vitro model is used to study the interaction between E. histolytica and colonic mucins.nnnMETHODSnSecretory mucins from the colonic adenocarcinoma cell line LS174T were collected and their functions assessed by their ability to inhibit amebic adherence to target cells and killing. The cytoprotective effect of mucus against E. histolytica cytolysis of LS174T monolayers was studied at 37 degrees C.nnnRESULTSnSepharose 4B column chromatography, metabolic labeling with [3H]glucosamine, cesium chloride density gradient centrifugation, and amino acid and carbohydrate compositional analysis revealed that LS174T cell mucins were typical of native colonic mucins. Mucin O-linked oligosaccharides bound to and inhibited the adherence of amebae to Chinese hamster ovary cells. E. histolytica killing of Chinese hamster ovary cell monolayers occurred rapidly, whereas killing of LS174T monolayers with an intact mucus layer was significantly retarded.nnnCONCLUSIONSnOur results show that colonic mucins serve as the first line of host defense against amebic invasion and provide a useful model to study pathogen-mucin interactions.

Interleukin (IL)-2, IL-4, and Tumor Necrosis Factor-α Responses during Entamoeba histolytica Liver Abscess Development in Gerbils

To determine cytokine production patterns during hepatic amebiasis, gerbils were infected with Entamoeba histolytica in the liver. Then spleen and hepatic lymph node cell proliferation and interleukin (IL)-2 (a Th1 marker), IL-4 (a Th2 marker), and tumor necrosis factor-alpha (TNF-alpha) production in response to concanavalin A and amebic antigen in vitro were quantified. Early abscess development (day 5 after inoculation) coincided with IL-2, IL-4, and low TNF-alpha production and strong lymphoproliferative responses, whereas suppression of IL production and lymphoproliferation occurred during acute disease (day 20). Proliferative responses and IL-2 production increased at days 30 and 60 after inoculation, but IL-4 levels remained low. Animals drug-treated at day 20 after inoculation demonstrated high IL-2 and low IL-4 production and resistance to reinfection. While acute hepatic amebiasis in gerbils is accompanied by transient immunosuppression, late infection and resistance to reinfection are associated with IL-2 production but low IL-4 and TNF-alpha production (Th-1-like response).

The human prostanoid DP receptor stimulates mucin secretion in LS174T cells

This study demonstrates the localization of the prostaglandin (PG)D2 receptor (DP) within the mucous‐secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase‐polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD2 and the highly selective DP‐specific agonist L‐644,698 ((4‐(3‐(3‐(3‐hydroxyoctyl)‐4‐oxo‐2‐thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE2 competed for [3H]‐PGD2‐specific binding to LS174T cell membranes (Ki values of 0.4u2003nM and 7u2003nM, respectively). The DP‐specific agonists PGD2, PGJ2, BW245C (5‐(6‐carboxyhexyl)‐1‐(3‐cyclohexyl‐3‐hydroxypropylhydantoin)), and L‐644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC50 values: 45–90u2003nM) and demonstrated the expected rank order of potency. PGE2 also elicited cyclic AMP production in this cell line (EC50 value: 162u2003nM). The activation of cyclic AMP production by PGD2 and L‐644,698, but not PGE2, was inhibited by the selective DP antagonist BW A868C. Thus, PGD2 and L‐644,698 act through hDP in LS174T cells. PGD2, L‐644,698 and PGE2 (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration‐dependent manner (EC50<50u2003nM). However, BW A868C effectively antagonized only the mucin secretion mediated by PGD2 and L‐644,698 and not PGE2. These data support a role for the DP receptor in the regulation of mucous secretion.

Construction and immunogenicity of a codon-optimized Entamoeba histolytica Gal-lectin-based DNA vaccine

Invasive amebiasis caused by Entamoeba histolytica is the third leading parasitic cause of mortality, and there are no vaccines available to help control the disease. The galactose-adherence lectin (Gal-lectin) is the parasites major molecule allowing it to adhere to colonic mucin for colonization and to target cells for tissue destruction. It is immunodominant and is regarded as the most promising candidate molecule to be included in a subunit vaccine against amebiasis. In this study, we are reporting the construction of a codon-optimized DNA vaccine encoding a portion of the Gal-lectin heavy subunit that includes the carbohydrate recognition domain (CRD), and its in vivo testing in mice. The vaccine stimulated a Th1-type Gal-lectin-specific cellular immune response as well as the development of serum antibodies that recognized a recombinant portion of the heavy subunit, and that inhibited the adherence of trophozoites to target cells in vitro.

A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages

The cysteine‐rich region of the 170‐kDa subunit galactose‐adherence lectin (Gal‐lectin) of Entamoeba histolytica is a subunit vaccine candidate and a protective antigen in the gerbil model of amebiasis. Macrophage‐mediated immunity is important for protection against E.u2009histolytica and is activated by Th1 cytokines. As Th1 differentiation is promoted by IL‐12, we investigated what portion of the Gal‐lectin could stimulate IL‐12 in human THP‐1 macrophages. Native Gal‐lactin stimulated IL‐12 p40u2009/u2009p35 mRNA expression in a dose‐ and time‐dependent manner as measured by reverse transcriptase‐PCR. Human immune serum and Gal‐lectin mAb inhibition studies identified amino acids (aa) 596u2009–u2009998 as immunogenic and containing the IL‐12 inducing domain. IFN‐γ priming augmented Gal‐lectin‐induced IL‐12 mRNA expression independent of TNF‐α and IL‐1β, and was required for IL‐12 p70 protein production from macrophages and human peripheral blood mononuclear cells. Gal‐lectin plus IFN‐γ stimulated IL‐12 p40 and p35 gene transcription with stable mRNA transcripts and a differential requirement for protein synthesis. These results suggest that aa 596u2009–u2009998 of the Gal‐lectin can confer Th1‐mediated protection against amebiasis through IL‐12 induction.